Many bacteria and many pathogenic bacteria in particular, are flagellated. The flagella and their action mechanism in the case of the E. coli species of bacteria have been precisely investigated. The flagella propel the bacteria, a process which is usually controlled by chemotaxis and enables them to find favorable living conditions. Repellants are avoided and attractants are followed by occasional statistical changes in direction (tumbling) after straight motions of controlled length. The control is based on concentration gradients of the attractants or repellants. Successive stimulus responses during the straight motion cause these straight motions to be continued if the conditions are favorable or to be discontinued, and a new direction tried, if the development of the concentrations is unfavorable. One percent concentration changes in a concentration range of more than five orders of magnitude can be perceived.
The rapid detection of pathogenic bacteria is important in food monitoring, in the search for sources of infection, and also to identify the type of infection contracted by a patient in order to combat the cause.
Salmonella is a genus of flagellated bacteria that belongs to the family of Enterobacteriaceae and is closely related to the Escherichia genus (usually also flagellated). According to the latest consensus only two species belong to the Salmonella genus, namely S. enterica and S. bongori, the former being subdivided into six sub-species with 2500 serovars nowadays. Most of the Salmonella species are pathogenic for humans and animals and may cause mild, but often severe, typhoidal or paratyphoidal bowel infections. They can survive for prolonged periods of time outside the human or animal organism (e.g., in dried feces demonstrably for 2.5 years), but are destroyed at 55° Celsius in one hour, at 60° Celsius in half an hour, making it relatively simple to disinfect equipment and food.
Salmonella infection occurs through oral ingestion, due to poor hygiene, infected water, or infected food. Salmonella infections are notifiable if they occur either endemically, or in places where nursing care is provided or food is produced.
The detection of Salmonella in the stool of patients, and also in food, is by nature always urgent. Known methods are slow or expensive. For example, they generally take at least two days, and usually three to five days.
A method which is both inexpensive and fast is urgently required. It should preferably provide a definitive result on the day after the sample was taken, at the latest. For food it is additionally desirable to be able to process many samples simultaneously without the analysis time and cost increasing significantly.
The conventional identification of microorganisms is usually based on standard methods that include of a series of consecutive culturing steps. Suitable selective broths or agar media are used, depending on the microorganism to be identified and on the particular application. After culturing, the actual identification of the individual colonies is performed either biochemically using the so-called “API test” or serologically by latex agglutination. These methods provide results after two days at the earliest, but sometimes may require five days. Two commonly used known methods will be briefly described.
To identify Salmonella in stool samples, a pea-sized stool sample is incubated overnight in ten milliliters of selective broth at 37° Celsius. Strongly selective methods must always be used here since stool samples always contain E. coli in large quantities also. The selective culture media used include selenite broth (enriches Salmonella, inhibits Escherichia), Rappaport-Vassiliadis broth (RVS, inhibits E. coli, but cannot be used for S. typhi or S. paratyphi), tetrathionate broth or Müller-Kauffmann tetrathionate broth with novobiocin (MKTTn, also inhibits E. coli). After this initial culture step, one milliliter of the liquid culture is plated on selective agar (e.g., XLD agar). After incubating overnight at 37° Celsius, the individual colonies obtained are characterized either biochemically (by the so-called “API test”), mass spectrometrically or serologically by agglutination. Characterization using “API test” requires a further 24-hour incubation of the microorganisms at 37° Celsius. With agglutination or mass spectrometry the result can be read off more or less directly. However, identifying Salmonella by the standard method takes at least two to three days.
In most countries, official standard methods exist for detecting Salmonella in food. In Germany, for instance, they are described in § 64 “Collection of Official Analytical Methods (ASU) according to German Food Law (LFGB: Lebensmittel-, Bedarfsgegenstände- and Futtermittelgesetzbuch)”. As an example, the L 00.00-20 method used to detect Salmonella will be described below. The usual detection procedure is to transfer 10 to 25 grams of the food under investigation to a non-selective pre-enrichment broth (buffered peptone water) in order to reactivate and, where necessary, propagate any Salmonella present (peptone water makes it possible to resuscitate sublethally damaged Salmonella). After incubating for 20 hours at 37° Celsius, two main enrichment cultures are started from the pre-enrichment culture. The main enrichment cultures contain selective media (Müller-Kauffmann Tetrathionate Broth (MKTTn) and Rappaport-Vassiliadis Broth (RVS)) and are inoculated with 0.1 to 1 milliliter of pre-enrichment culture, depending on the broth. MKTTn cultures are incubated at 37° Celsius and RVS cultures at 41.5° Celsius for 24 hours. A smear from each of these main enrichment cultures is made on an XLD agar and a second selective agar (a Rambach agar, for example). After incubating the plates for 24 hours at 37° Celsius, suspect colonies are investigated for Salmonella. If no suspect colonies have grown, the result is negative for Salmonella. To characterize possible suspect colonies further, they are subcultured on a CASO agar (24 hours, 37° Celsius). Salmonella is then detected using either “API test” or latex agglutination. This standard method for Salmonella takes four to five days.
Detection methods from molecular biology, which have major advantages over these conventional methods, have been known for a number of years. In the food sector, to identify Salmonella a method of identifying many microorganisms by DNA analysis after PCR amplification (polymerase chain reaction) is disclosed in U.S. Published Patent Application 2006 177 824 A1. In contrast to the standard methods of culturing, this method can provide a result after only one to two days and thus saves valuable time. Its disadvantage includes the relatively high cost per culture, taking into account the fact that food inspections usually involve many samples each time (often a few hundred). Furthermore PCR is prone to interference, depending on the sample. Extensive positive and negative controls have to be carried out to validate the results.
A further method from molecular biology is based on a mass spectrometric analysis of microbe-specific molecular cell components. This method is superior to conventional methods in terms of specificity (true-negative rate), sensitivity (true-positive rate), other error rates, and particularly in terms of cost and analytical speed.
The process of generating mass spectra of the components of the cultured microbes usually starts with a cleanly isolated colony on a solid, usually gelatinous nutrient medium or a centrifuge sediment (pellet) from a liquid nutrient medium. A tiny quantity of microbes is transferred from the selected colony or sediment to the mass spectrometric sample support, using a small swab, such as a wooden tooth pick. An acidified solution of a conventional matrix substance is then sprinkled onto this sample, the matrix substance being used for subsequent ionization of the microbe components by matrix-assisted laser desorption (MALDI). The acid of the matrix solution attacks the cell walls and weakens them; the organic solvent penetrates the microbial cells, causing them to burst by osmotic pressure, and releases the soluble proteins. The sample is then dried by evaporating the solvent, which causes the dissolved matrix material to crystallize. The soluble proteins and, to a lesser extent, other substances of the cell also are thus embedded into the matrix crystals.
Instead of transferring whole microbes by swabs, the microbes cleaned by washing can also be disintegrated in vitro, in a centrifuge tube, for example, where strong acids can be used to destroy the microbial cell wall. Centrifuging separates the insoluble components such as cell walls so that they can no longer interfere with the mass spectrometric analysis. Around one microliter of the solution is applied to the mass spectrometric sample support and dried there. This analysis sample is then coated with a suitable matrix solution and dried again. During the drying process, protein molecules are incorporated into the small matrix crystals which form. These disintegration produce mass spectra which are practically identical to those of the usual preparation on sample supports, but are cleaner; they exhibit less interfering background and are therefore better suited to detecting pathogens, in mixtures with other microbes also.
The sample preparations dried on sample supports, i.e., the matrix crystals with the embedded protein molecules, are bombarded with pulsed UV laser light in a mass spectrometer, thus creating ions of the protein molecules that can then be measured, with separation according to the mass of the ions in the mass spectrometer. This type of ionization by matrix-assisted laser desorption is usually referred to as Matrix-Assisted Laser Desorption and Ionization (MALDI). It is preferable to use MALDI time-of-flight mass spectrometers for this purpose. Several types of crystalline organic acids can be used as matrix substances: HCCA(α-cyano-4-hydroxycinnamic acid), for example.
Nowadays, the mass spectra of the microbe proteins are scanned in the linear mode of these time-of-flight mass spectrometers, i.e., without using an energy-focusing reflector, because this mode gives a particularly high detection sensitivity, although the mass resolution and the mass trueness of the spectra from time-of-flight mass spectrometers in reflector mode is considerably better. The lack of reproducibility of the desorption and ionization processes for the generation of the ions means that the masses of the individual mass signals shift slightly from spectrum to spectrum. These shifts in the mass scales of the repeat spectra can be readjusted with respect to each other using a method described in U.S. Pat. No. 7,391,017, before homogeneous groups of repeat spectra are combined to form a sum spectrum, which is then used as a reference spectrum or sample spectrum. The mass scales of sample and reference spectra can also be adjusted with respect to each other by this mass scale adjustment program. This means that smaller mass tolerance intervals can be used for the determination of matching mass signals during the similarity analysis, which is crucial for a good identification, even if it takes some time.
The mass spectrum of a microbial isolate is the frequency profile of the ions of the soluble cell components, separated according to mass The ions are predominantly protein ions. The mass spectra are usually acquired in the mass range from 2,000 to 20,000 daltons; the most useful information for identifications is found in the mass range from around 3,000 daltons to 15,000 daltons. Each laser light pulse produces a single mass spectrum, which is measured in less than 100 microseconds but contains the signals of only a few hundred to a few thousand ions. In order to obtain more reliable and less noisy mass spectra, a few tens to a few thousands of these individual mass spectra are added together to form a sum mass spectrum. The individual mass spectra can preferably originate from different parts of the sample preparation or even from different sample preparations. The term “mass spectrum of a microbe”, or more simply “microbe spectrum”, refers to this sum mass spectrum. The acquisition of such a microbe spectrum takes only a few seconds due to the high laser bombardment rates (currently up to two kilohertz). A sample support plate with 48 or even 384 samples may be automatically measured in less than half an hour.
The profile of the proteins reproduced by each of these microbe spectra is characteristic of the species of microbe in question because each species produces its own, genetically predetermined proteins, each having their own characteristic masses. The abundances of the individual proteins in the microbes, in as much as they can be measured mass spectrometrically, are also genetically predetermined to a large extent because their production is controlled by other proteins, and they depend only slightly on the nutrient medium or the degree of maturity of the colony. The protein profiles are characteristic of the microbes in the same way that fingerprints are characteristic of humans. This makes it possible to identify the microbes by a similarity analysis with reference spectra from a reference library.
The spectra acquired are evaluated with programs provided by the manufacturers of the mass spectrometers. These programs are based on similarity analyses between a measured microbe spectrum and reference mass spectra from specially validated spectral libraries. This is done by calculating a similarity index score for each reference spectrum. If the highest index score exceeds a specified similarity threshold, it is clear proof that the microbe species belonging to the corresponding reference spectrum is present. There are special similarity thresholds for the assignment of microbes to families, genera or species.
It must be emphasized that the mass spectrometric method has so far been mainly used for the identification of unknown bacteria. Bacteria isolates from well-separated colonies on agar plates have usually been used for the sample preparation. The identification of two, at best three, bacterial species in a mixture of these two or three species is disclosed in German Patent DE 10 2009 007 266.7. A great strength of the mass spectrometric method which has not been utilized so far is its ability to detect the presence of a target bacterial species in somewhat more complex mixtures of five, ten or more bacterial species unambiguously and reliably if the signature of this target species is still detectable in the mass spectrum of the mixture at all. It is not necessary, and often not possible, to identify all the bacteria of the mixture. Even the absence of a target bacterial species can be identified with certainty if a signal of the target bacterial species which is definitely to be expected is missing in at least one location in the mixture spectrum. Special evaluation programs enable the presence or absence of target bacteria to be detected unambiguously and reliably even if they amount to only one to ten percent of the mixture, depending on the complexity of the mixture. This type of detection of a target bacterial species in mixtures has not been elucidated so far, especially since the evaluation programs commercially available to date are not designed for this task.